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CP-B (CP-52731)
CP-B (CP-52731)
規格:
價格:
編號:B235382
品牌:Mingzhoubio

標準菌株
定量菌液
DNA
RNA

規格:
凍干粉
斜面
甘油
平板


產品名稱
CP-B (CP-52731)
商品貨號
B235382
Organism
Homo sapiens, human
Tissue
Esophagus; epithelium
培養基
Cell Type
Epithelial cells immortalized with hTERT
Product Format
frozen
Morphology
Epithelial-like
Culture Properties
Adherent
Biosafety Level
2

Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country.

Disease
High-grade dysplasia, Barrett's esophagus
Age
Adult
Gender
Male
Applications
These well-characterized pre-malignant cultures represent a unique opportunity to study factors that are important in both cancer progression and hTERT immortalization.
Storage Conditions
Liquid nitrogen vapor phase
Karyotype
This is a hypodiploid cell line of male origin with the following derivative chromosomes consistently present at different passages: der(1)t(1;17)(q42?;q21), add(8)(p11.2), der(9)t(9;14)(q10;q10), add(12)(q13), add(15)(q24.3), add(17)(p11.2), del(19)(p13.1), del(21)(q22.1). It should be noted that the tetraploid population was essentially a duplicate of the hypodiploid population and may range from around 18% at lower passages to as high as 50% at higher passages.
Images
Derivation
The Barrett's esophagus cell line, CP-B (also identified as CP52731 or ChTERT), was derived from an endoscopic biopsy specimen obtained from a region of high-grade dysplasia and transduced with the retroviral expression vector, pLXSN-hTERT, to create an immortalized cell line.
Clinical Data
male
Antigen Expression
Antigen expression: This cell line is positive for epithelial marker pan-cytokeratin (immunocytochemistry)(verified at ATCC), and negative for gastric mucin (CLH2) (immunocytochemistry)(verified at ATCC).
Comments

This cell line shows increased telomerase activity and long telomeres of about 12 kb as assessed by terminal restriction fragment lengths (TRF) analysis. Morphologically, the cells are similar to early passage cultures: smaller cells with a larger nucleus to cytoplasm ratio. Genetic instability studies using flow cytometry and FISH reveal the retention of elevated tetraploidy (G2/tetraploidy) in the hTERT-immortalized cells, similar to the non-transduced parental cells.


Complete Growth Medium
The base medium for this cell line is MCDB-153. For complete growth medium, add the following components to the base medium:
  • 0.4 µg/ml hydrocortisone
  • 20 ng/ml recombinant human epidermal growth factor
  • 8.4 µg/L cholera toxin
  • 20 mg/L adenine
  • 140 µg/ml bovine pituitary extract
  • 1x ITS Supplement (Sigma; I1884) 
  • 4 mM glutamine
  • fetal bovine serum to a final concentration of 5%
To prepare Cholera toxin (Stock 100 µg/mL): 0.5 mg Cholera toxin (Sigma C8052) + 5 mL dH20. Add 84 µL of this 100 µg/mL stock solution to 1L of MCDB-153 base medium. Do not filter the complete medium.
Subculturing
Volumes used in this protocol are for 75 cm2 flasks; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes.
  1. Remove and discard culture medium.
  2. Add 2.0 to 3.0 mL of 0.25% (w/v) Trypsin - 0.53 mM EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).
    Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37.0°C to facilitate dispersal.
  3. Add 6.0 to 8.0 mL of complete growth medium and aspirate cells by gently pipetting.
  4. Transfer cell suspension to a centrifuge tube and spin at approximately 125 x g for 5 to 10 minutes. Discard supernatant.
  5. Resuspend the cell pellet in fresh growth medium. Add appropriate aliquots of the cell suspension to new culture vessels. An inoculum of 2 X 104 to 3 X 104 viable cells/cm2 is recommended.
  6. Incubate cultures at 37°C. Subculture when cells reach a concentration between 1 X 105 and 2 X 105 cells/cm2.
Subcultivation ratio: A subcultivation ratio of 1:3 to 1:6 is recommended.
Medium renewal: every 3 to 4 days
Note: For more information on enzymatic dissociation and subculturing of cell lines consult Chapter 13 in Culture Of Aminal Cells: A Manual of Basic Techniques by R. Ian Freshney, 5th edition, published by Wiley-Liss, N.Y., 2005.
Cryopreservation
Freeze Medium: RPMI-1640 Medium, 80%; fetal bovine serum, 10%; DMSO, 10%
Storage Temperature: liquid nitrogen vapor phase
Culture Conditions
Temperature: 37°C
Atmosphere: air, 95%; carbon dioxide (CO2), 5%
STR Profile
CSF1PO: 8, 12
D13S317: 8, 12
D16S539: 10, 13
D5S818: 11, 12
D7S820: 11, 12
THO1: 8, 9
TPOX: 8, 9
vWA: 18, 20
Amelogenin: XY
Population Doubling Level (PDL)
As part of our quality control, we have tested this cell line for its ability to grow for a minimum of 15 population doublings after recovery from cryopreservation. We have also compared its karyotype, telomerase expression level, growth rate, morphology and tissue-specific markers when first recovered from cryopreservation with that of cells at 10+ population doublings to ensure that there is no change in these parameters and that the cells are capable of extended proliferation.
Population Doubling Time
about 34 hours
Name of Depositor
B Reid
Year of Origin
November 1994
References

Palanca-Wessels MC, et al. Genetic Analysis of Long-term Barrett's Esophagus Epithelial Cultures Exhibiting Cytogenetic and Ploidy Abnormalities. Gastroentrology 114:114-295, 1998. PubMed: 9453489

Palanca-Wessels MC, et al. Extended lifespan of Barrett's esophagus epithelium transduced with the human telomerase catalytic subunit: a useful in vitro model. Carcinogenesis 24(7): 1183-1190, 2003. PubMed: 12807723

Barrett MT, et al. Molecular Phenotype of Spontaneously Arising 4N (G2-Tetraploid) Intermediates of Neoplastic Progression in Barrett's Esophagus. Cancer Res. 63: 4211-4217, 2003. PubMed: 12874028

Maley CC, et al. Genetic clonal diversity predicts progression to esophageal adenocarcinoma. Nat. Genet. 38(4): 468-473, 2006. PubMed: 16565718

Freshney RI. Culture of Animal Cells: A Manual of Basic Technique, 5th edition. New York: Wiley Liss; 2005. For more information on enzymatic dissociation and subculturing of cell lines see Chapter 13.

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